Epidermal Growth Factor Receptor (EGFR) and the de2-7 EGFR as Targets for Therapy
Immunotherapeutic treatment of cancer has the advantage over traditional therapies such as surgery, radiotherapy and chemotherapy, in that there can be a high specificity for the disease target. Tumour specific mAbs can be used to target cancer cells, creating a need to identify and locate tumour-associated antigens as potential targets. The overexpression of growth factor receptors such as EGFR, IL-2 receptor and p185 HER2 is often associated with tumours such as lung, breast, head and neck, and ovarian tumours.
The EGFR belongs to a family of tyrosine kinase growth factor receptor proteins. The EGFR has long been the subject of investigation, and recently there have been successful structure determination studies performed of the extracellular domains (Ogiso H et al. Cell 2002, 110:775-787; Garrett T P et al. Cell 2002, 110:763-773; Ferguson K M et al Cell 2003, 11:507) and intracellular kinase domain (Stamos J et al J. Biol. Chem. 2002, 277:46265-46272). This has provided vital information into the behaviour of the receptor and its ligands. The EGFR is a cell surface associated molecule, which is activated through binding of highly specific ligand, such as EGF and transforming growth factor alpha (TGF α). After ligand binding, the receptor dimerizes, which results in phosphorylation of the intra-cellular tyrosine kinase region. This leads to downstream signaling, activating a cascade of responses resulting in cell growth and proliferation. Given that tumour cells, unlike normal cells, are dependent on the EGFR for function, and because of the range of possibilities of inhibiting EGFR's regulatory control of proliferation and differentiation in cells, the receptor is a common target for therapy. The EGFR is normally expressed in the liver and skin, with increased activity often found in solid tumours, such as head and neck, colorectal, pancreas, glioma, bladder and lung, thus making it a useful prognostic marker. Overexpression of the EGFR is often accompanied by increased TGF α production effecting an autocrine loop growth advantage to the tumour. Furthermore, it was found that the EGFR gene amplification and rearrangement which is observed in some tumours, is often associated with the occurrence of mutant forms of the EGFR (Libermann T A, et al Nature 1985, 313:144-147; Wong A J, Proc Natl Acad Sci USA 1992, 89:2965-2969; Frederick L, et al Cancer Res 2000, 60:1383-1387). One of the most common mutants is the EGFR variant (EGFR vIII or de2-7EGFR). The de2-7EGFR has an in-frame deletion of 801 base pairs, corresponding to an over-expression of transcripts missing exons 2-7, and a sizeable deletion of amino acid residues 6-273 in the extracellular domain, with a novel glycine inserted at the splice site (Wong A J et al. Proc Natl Acad Sci USA 1992, 89:2965-2969; Sugawa N. et al Proc Natl Acad Sci USA 1990, 87:8602-8606; Yamazaki H. et al Jpn J Cancer Res 1990, 81:773-779; Ekstrand A J et al Proc Natl Acad Sci USA 1992, 89:4309-4313). This truncated form of the EGFR is not dependent on ligand binding, and is constitutively active. The de2-7EGFR is expressed in a large fraction (>50%) of malignant gliomas and there are also reports linking the de2-7EGFR with breast (27%), ovarian, prostate and lung carcinomas (17%) (Wong A J, et al Proc Natl Acad Sci USA 1992, 89:2965-2969; Garcia d P et al Cancer Res 1993, 53:3217-3220; Wikstrand C J et al Cancer Res 1995, 55:3140-3148; Moscatello D K et al Cancer Res 1995, 55:5536-5539).
Anti-EGFR Antibodies
Many studies have focused on the production of antibodies to the extracellular region of the EGFR. The mAbs generated mediate their anti-tumour activity primarily by blocking ligand binding and also the disruption of signaling. There were several mAbs initially developed by Peng et al. 1996 (Peng D et al Cancer Res 1996, 56:3666-3669) and Mendelson et al. 1997 (. Mendelsohn J Clin Cancer Res 1997, 3:2703-2707) to specifically recognize the EGFR. Mabs 425, 528 IgG2a and 225 IgG1 were used to treat patients with head and neck squamous cell carcinoma (Sturgis E M, et al Otolaryngol. Head Neck Surg 1994, 111:633-643). Experimental work, including radiolabelling, has shown the mAb 425 to be an effective inhibitor of tumour growth including gliomas (Rodeck U et al J Cell Biochem 1987, 35:315-320; Brady L W et al Int J Radiat Oncol Biol Phys 1991, 22:225-230; Faillot T et al Neurosurgery 1996, 39:478-483). The IMC-C225 mAb specifically recognizes the EGFR, and has much potential in the treatment of cancers such as head and neck, colorectal, pancreas and lung. The mAb255 up-regulates p27 K1P1 and induces G1 arrest in a prostatic cancer cell line. Subsequently, a chimeric version (ERBITUX™ (Imclone Systems, NY) IMC-C225) of the mouse 225 antibody was developed to extend its therapeutic capability. The IMC-C225 has increased binding affinity for the EGFR and is more effective in reducing xenograft growth in mice. Both mouse and chimeric antibodies are even more effective when given in combination therapy with radiation (Robert F et al J Clin Oncol 2001, 19:3234-3243) or chemotherapy (Shin D M et al Clin. Cancer Res 2001, 7:1204-1213). The therapeutic mechanism of action of the IMC-C225 appears to include an efficient receptor blocking function and a capacity for ADCC. IMC-225 can reduce tumour size in patients. Large doses of IMC-C225 are required to saturate the liver and skin binding sites and the adverse effects are primarily acneform rash and pruitis. Clinical trials have shown partial response rates of tumour growth in patients of between 11% and 22% when combined with cisplatin. The preclinical and clinical progress of this antibody is covered in reviews by Baselga et al. [49] and Mendelsohn et al. (Baselga J et al J Clin Oncol 2000, 18:904-914; Mendelsohn J J. Clin. Oncol. 2002, 20 Suppl 1:1 S-13S).
The mAb R3 was raised against the EGFR and was initially developed for use in radioimmunotherapy (Waterfield M D, et al. J. Cell Biochem. 1982, 20:149-161; Ramos-Suzarte M, et al. J. Nucl. Med. 1999, 40:768-775). Both chimeric and humanized forms of R3 have been produced and tested in African Green monkeys. The humanized version of R3 retained the same binding affinity of the mouse antibody, and was found to be 2-fold less immunogenic than the chimeric antibody. Preclinical studies of xenografts in mice using technetium-labeled mouse and humanized mAbs, showed a greater potential as a diagnostic tool with the humanized version than the murine. The rat anti-EGFR mAb, ICR62, effectively competes for ligand binding and eradicates human tumour xenografts (squamous cell carcinomas) in mice. Phase I clinical trials reported the antibody was administered safely to patients with squamous cell carcinomas, and it has since been used to investigate the signaling pathways of growth factor receptors and their ligands in head and neck squamous cell carcinoma cell lines (O-charoenrat P et al Clin. Exp. Metastasis 2000, 18:155-161; O-charoenrat P et al. Int. J. Cancer 2000, 86: 307-317; O-charoenrat P et al Oral Oncol. 2002, 38:627-640).
The anti-EGFR mAb 108.4 exhibited an anti-tumour effect that was enhanced when combined with cisplatin (Aboud-Pirak E et al J Natl Cancer Inst 1988, 80:1605-1611). The same result occurred with the Fab fragment alone, which suggests the mechanism does not rely on the interaction of the Fc with the host complement system. In another example, the potential of combination therapy was investigated with the mAb RG 83852, with respect to understanding the underlying mechanism between antibody and receptor (Perez-Soler R et al J Clin Oncol 1994, 12:730-739). It was suggested that up-regulation of the EGFR by mAb RG 83852, increased the tyrosine kinase activity of the receptor within the tumour, thus increasing its susceptibility to chemotherapy. Targeted irradiation by monoclonal antibodies is another approach to cancer treatment. A number of studies on the effect of radiolabelling several anti-EGFR antibodies in the treatment of glioma has been undertaken by Kalofonos (Kalofonos H P et al J Nucl. Med 1989, 30:1636-1645). These studies reported good targeting and minimal toxicity. The humanized mAb ENID 72000 which blocks ligand binding in the EGFR is currently undergoing clinical trials (Bier H et al Cancer Chemother. Pharmacol. 2001, 47:519-524). Lastly, the fully human antibody ABX-EGF derived from transgenic mice also effectively targets the EGFR (Yang X D et al Crit. Rev. Oncol. Hematol. 2001, 38:17-23).
Anti de2-7 EGFR Antibodies
The wild-type EGFR is expressed on most epithelial cells; so a drawback to therapeutically targeting the receptor is the side effect of toxicity to normal tissue as well as cancer cells. Additionally, such antibodies when conjugated with radio-isotypes or cytotoxic agents may cause potential harm to normal tissue. Ideally it would be advantageous to preferentially target the EGFR on cancer cells. The de2-7EGFR is an attractive therapeutic target because in adults it is highly specific to cancer cells. There have been studies performed with antibodies against the de2-7EGFR where the inhibition of cell growth in cancer cell lines has been shown. The mAbs 528 (Sturgis E M et al Otolaryngol. Head Neck Surg 1994, 111:633-643; Masui H et al Cancer Res 1984, 44:1002-1007) and 425 (described above) bind to both the de2-7EGFR and EGFR. The unique sequence of the de2-7EGFR generated by the insertion of a glycine at the splice site, creates a novel epitope, located near the N-terminus of the extra-cellular region (Humphrey P A et al Proc Natl Acad Sci USA 1990, 87:4207-4211; Lorimer I A et al Clin Cancer Res 1995, 1:859-864). Several antibodies, specific for the fusion junction have been produced, including mAb Y10 (Wikstrand C J et al Cancer Res. 1995, 55:3140-3148; Okamoto S et al. Br. J Cancer 1996, 73:1366-1372; Sampson J H et al. Proc Natl Acad Sci USA 2000, 97:7503-7508). This antibody, which was used effectively to treat brain tumour xenografts in mice, functions mechanistically by reducing cell growth, and also showed capacity for ADCC and CDC. Antibodies generated against peptides of the sequence specific for the fusion junction include the MRI, an Fv fragment generated by phage display (Lorimer I A et al Proc Natl Acad Sci USA 1996, 93:14815-14820). The Fv has the ability to infiltrate solid tumours, and has been used to deliver an immunotoxin. Several antibodies targeting the fusion junction of de2-7EGFR have been radiolabelled: these include L8A4, DH8.3 and Ua30:2 (Reist C J et al Cancer Res 1997 57:1510-1515; Hills D et al Int J Cancer 1995, 63:537-543; Ohman L et al Tumour Biol 2002, 23:61-69). The radiolabelled DH8.3 antibody recognises the de2-7EGFR, but not the normal EGFR, and reduces tumour size in nude mice.
The Murine Anti-EGFR Antibody mAb-806
The murine monoclonal antibody mAb-806 (class IgG2b) has been shown to bind de2-7EGFR, but not normally expressed wild-type EGFR (Patent Application WO 02/092771; Johns T G et al Int J Cancer 2002, 98:398-408). Although mAb-806 does not react with the normal wild type receptor, it does recognize a proportion (˜10%) of wild type EGFR on tumour cells containing amplified EGFR genes (Johns T G et al. Int J Cancer 2002, 98:398-408; Luwor R B et al Cancer Res. 2001, 61:5355-5361). The ability of mAb-806 to target both de2-7EGFR and amplified wild-type EGFR, both of which occur with notable frequency in tumours, should confer added effectiveness for mAb-806 as a therapeutic agent.
MAb-806 differs from other antibodies that target the de2-7EGFR, in that it does not recognize the unique fusion junction of de2-7EGFR (Wong A J, et al. Proc Natl Acad Sci USA 1992, 89:2965-2969: Sugawa N. et al. Proc Natl Acad Sci USA 1990, 87:8602-8606; Yamazaki H, et al. Jpn. J Cancer Res 1990, 81:773-779: Ekstrand A J, et al. Proc Natl Acad Sci USA 1992, 89:4309-4313). The binding epitope of mAb-806 exists in both the wild type and truncated de2-7EGFR. Given the ability of mAb-806 to bind both the de2-7EGFR and the amplified EGFR, and its absence of binding to normally expressed wild-type receptor, it has been assumed that the epitope is conformationally dependent. Many antibodies against the wild-type EGFR in clinical development function by blocking ligand binding. This would not appear to be the mechanism of action of mAb-806 because of the characteristics of binding both with the non-ligand binding de2-7EGFR and the amplified EGFR, and the absence of binding with the normal receptor. This indicates that mAb-806 does not interfere with ligand binding or dimerization.
The mAb-806 antibody binds to de2-7EGFR expressed on the U87MG.de2-7EGFR cell line, but not to the parental cell line (U87MG) which contains unamplified wild-type EGFR. In comparing the efficacy of the mAb-806 with the DH8.3 mAb, it was established that mAb-806 was more efficient in tumour targeting, and had stronger binding than the DH8.3 (EGFR (Johns T G et al Int J Cancer 2002, 98:398-408). MAb-806 was shown to inhibit the growth of mice xenografts in a dose dependent manner using the A431 cell line containing amplified EGFR (Johns T G et al Int J. Cancer 2002, 98:398-408), as well as U87MG.de2-7EGFR. Again growth inhibition was not observed in the parental U87MG xenografts. Significantly, reduced tumour growth has also been shown for intercranial xenografted glioblastomas upon application of mAb-806 to U87MG.de2-7EGFR, LN-Z308. de2-7EGFR, and A1207.de2-7EGFR xenograts (all expressing de2-7EGFR) (Mishima K et al Cancer Res. 2001, 61:5349-5354). No significant inhibition was observed in xenografts of the parental U87MG tumours, U87 MG.DK tumours (expressing kinase deficient de2-7EGFR), and only a small response occurs in the U87MG glioma. A reduction in angiogenesis and an increase in apoptosis occur concurrently with the reduction in tumour growth.
With its unique properties, the mAb-806 antibody is a promising therapeutic for the treatment of cancers such as head and neck cancer, and glioma. The development of a humanized form of mAb-806 will have a major effect on its efficacy. Such an antibody should avoid a HAMA response, improve its ability to recruit effector function and increase its half-life in circulation, thus greatly enhancing its clinical prospects.
Initially there were high expectations for the use of mAbs as therapeutic magic bullets, but it was soon realised that there are several major impediments limiting the clinical use of non-human antibodies. The administration of multiple doses of non-human mAbs generally provokes an unwanted immune response thus severely limiting their use as a therapeutic. The mouse antibody is recognized by the human immune system as a foreign protein resulting in an immune effect known as the human anti-mouse antibody response, i.e. the HAMA response. The HAMA response can result in neutralization of the antibody function and in serious allergic-like reactions.
Much of the HAMA response is directed against the antigen binding portion (Fab) and rarely the constant regions (Fc) of the antibody. Additional problems resulting from the clinical application of rodent mAbs are associated with the Fc regions. The human Fc binds to specialised Fc receptors, which help to maintain the antibodies in circulation. As a result, rodent mAbs have a shortened half-life, usually 1-3 days as compared with a week or more for human Ig. Another limitation is the reduced recruitment of a variety of effector functions initiated on binding of the Fc to the human Fc receptor. The binding to Fc receptors of specialized effector cells such as macrophages, monocytes and neutrophils, triggers the immune system leading to a response known as antibody-dependent cell-mediated cytolysis (ADCC). Fc receptors are also responsible for the triggering of the complement cascade (a group of interacting proteins) leading to the complement-dependent cytolysis response (CDC). This results in cell lysis and increases the effectiveness of antibodies to fight bacterial infection. The class of the constant domains predominantly controls the efficacy of the antibody in cell lysis.
There are different approaches that may be taken to overcome the immunogenicity of mouse mAbs, such as rapid infusion of antibody dose and the use of antibody fragments (e.g. single chain Fv (scFv) see Carter P Nat. Rev. Cancer 2001, 1:118-129; Hudson P et al Nature Med 2003, 9:129-134 and references therein). Alternatively, antibody engineering methods have been employed to reduce the HAMA response when whole IgGs are used for therapy.
This approach has the added potential advantages of increasing half-life and more effective recruitment of effector function. Such humanization methods are well known within the art and have for example been described in U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, 5,859,205, and 6,797,492 each incorporated herein by reference.
Human Antibodies
An alternative approach to overcoming the problem of immunogenicity in mAbs is the production of completely (fully) human antibodies. Phage display technology can be used to select a range of human antibodies binding specifically to the antigen using methods of affinity enrichment (McCafferty J et al Nature 1990, 348:552-554; Azzazy H M et al Clin. Biochem. 2002, 35:425-445). The bacteriophage is a virus that only infects bacteria, and reproduces in Escherichia coli. The phage display process involves the insertion of human genetic material into the phage genome. The filamentous phage system has the unique property where the structural and functional information of the ligand displayed on the phage surface (phenotype) is linked to the ligand's genetic information within the phage genome (genotype). Therefore, a library of Ig molecules can be generated and displayed on the surface of filamentous phage, and those showing binding affinities are selected. This method has the advantage of a very rapid simultaneous screening of many antibodies with high antigen affinity. It has also been used successfully in antibody humanizations by generating a combinatorial library including a set of potentially critical residues needed to preserve full binding avidity. The framework can then be optimised by random mutagenesis of the critical residues.
Transgenic Mice
Recently, an alternative approach to phage display methodology of producing human mAbs was developed where the human genes are inserted into the mouse DNA creating transgenic mice, capable of generating fully human protein sequences (for reviews of the methods involved, see references Little M et al Immunol. Today 2000, 21:364-370; Humphreys D P et al Curr. Opin. Drug Discov. Devel. 2001, 4:172-185; Ishida T et al Nippon Rinsho 2002, 60:439-444). Accordingly, these mice can produce human antibodies in response to immunization with a target antigen. The antibodies generated are effectively human and would not be expected to be rejected by the host immune system. The XenoMouse® produced by Abgenix contains approximately 80% of the human heavy chain genes, and a large number of light chain genes. Different strains of the mice have been produced containing different classes of antibodies capable of targeting a range of diseases (Yang X D et al Cancer Res 1999, 59:1236-1243; Davis C G et al Cancer Metastasis Rev 1999, 18:421-425). For example, ABX-MA1 is a fully human antibody which targets MCAM/MUC18 (a glycoprotein associated with tumour thickness and metastases in human melanoma cells in mice) and shows promise in the treatment of melanoma (Mills L et al Cancer Res 2002, 62:5106-5114). ABX-EGF targets the EGFR, and is currently in phase I/II clinical trial in the treatment of head and neck, non-small cell lung carcinoma, and colon cancer.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods and the recognition of the usefulness and application of antibodies in the diagnosis, treatment, and prevention of disease, it should be apparent that there still exists a need in the art for a preparation and use of humanized/fully human antibodies, particularly directed against the EGF receptor. There is a particular need for humanized/fully human antibodies which demonstrate reduced or absence of antibody immune response in humans and that recognize oncogenic or activated forms of EGFR as well as amplified or overexpressed forms of EGFR.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.